Recent data estimates 1.4 million transgender adults and 150,000 transgender adolescents live in the United States, many of whom are on cross-sex hormone therapy with estradiol or testosterone (T). National and international medical organizations recommend fertility preservation counseling prior to initiation of cross-sex hormone therapy, based on an assumption of fertility loss. However, the impact of long-term cross-sex hormone therapy on reproductive health is largely unknown, particularly in transgender men (female-to- male or FTM). The available human studies suggest ovarian changes from cross-sex T therapy, but are observational studies, with conflicting results. Moreover, there is a lack of data on the fecundity of T-treated transgender men, and there have been no studies that address the reversibility of T-induced changes after cessation of T for reproductive purposes. There are also no studies on future reproductive consequences of the treatment paradigm for transgender adolescents, in which GnRHa is initiated in the early peripubertal period to suppress further pubertal progression prior to transitioning directly to T therapy (GnRHa-T). None of the existing animal models that address the effect of androgens on reproductive function in females are directly applicable to the clinical paradigm of cross-sex T therapy in transgender men or GnRHa-T therapy in transgender adolescents. To address this knowledge gap, we have developed a mouse model to mimic T treatment for FTM gender transition. These mice manifest defects in ovarian architecture and have altered folliculogenesis. This model provides a powerful tool to clarify the effects of T therapy on reproductive phenotype and function, in a manner not possible in humans. The objective of the proposed studies is to use the FTM mouse model to investigate the effects of cross-sex T treatment on reproductive phenotype and function, and determine the reversibility of these effects following cessation of T. Our central hypothesis is that T therapy will adversely affect ovarian architecture and fertility, but that fertility can be recovered with cessation of T, without adverse reproductive effects in offspring. To test this hypothesis, we will examine the reversibility of postpubertal T administration in female mice, mimicking FTM gender transition, on reproductive phenotype (AIM 1), and examine fertility during and after cessation of long-term testosterone therapy, including reproductive phenotype in offspring (AIM 2). In an exploratory aim, we will examine the reproductive consequences of testosterone administration after pretreatment with peripubertal GnRHa, mimicking FTM cross-sex hormone therapy in adolescents, on reproductive phenotype and fertility (AIM 3). This proposal challenges the status quo of recommending fertility preservation prior to cross-sex T therapy, and will lay the foundation for further translational studies. Our long-term goal is to provide the necessary data for evidence-based fertility counseling of transgender men. Clarifying the effects and reversibility of cross-sex T therapy on the reproductive tract could lead to future paradigm shifts in clinical fertility care of transgender men.